We have shown that retinoid signaling is engaged during 11-deoxy-16,16-dimethyl PGE2 (DDM-PGE2) mediated cytoprotection against reactive oxygen species (ROS) induced necrotic/oncotic cell death. Proteomics analyses revealed that cytoprotection is associated with the increased synthesis of a select number of proteins, including retinol binding protein (RBP), actin, and glucose-regulated protein 78 (Grp78). We subsequently confirmed that all-trans-retinoic acid (aTRA) replicates DDM-PGE2-mediated cytoprotection in vitro, and more importantly, a single dose of aTRA (1 mg/kg, 6h pretreatment) completely protects mice from renal ischemia/reperfusion (I/R) injury. Furthermore, at this therapeutic dose, aTRA induces Nrf2-responsive antioxidant HO-1 and NQO1 genes, as well as nuclear retinoic acid receptors RAR?, RAR?2, RAR?2, and retinoid X receptors RXR? in the kidney. The revised application is designed to determine the molecular mechanisms by which aTRA affords cytoprotection in vitro, and the extent to which this mechanism(s) of cytoprotection is recapitulated in vivo. Our central hypothesis is that aTRA-induced cytoprotection is mediated by mechanisms similar to ischemic preconditioning. In Specific Aim 1 we propose to determine the ability of aTRA to offer cytoprotection in an in vitro model (human renal epithelial HK-2 cells) of hypoxia/reoxygenation injury, and to optimize protocols for aTRA-mediated cytoprotection in an in vivo ischemia/reperfuson model (IR). We will also ascertain whether the protective effects are mediated, at least in part, via the upregulation of anti-oxidant enzymes. The biological effects of retinoids are typically mediated via interaction with their cognate nuclear receptors, namely, retinoic acid receptors (RAR) and retinoid X receptors (RXR). The extent to which RAR and/or RXR participate in aTRA-mediated cytoprotection is not known, and Specific Aim 2 will determine, in both the in vitro and in vivo models of I/R, whether aTRA-mediated cytoprotection requires interaction with RAR and/or RXR. Specific Aims 1 and 2 are therefore designed to establish the recruitment of retinoid signaling as a potential therapeutic intervention in conditions where ROS play an important role in the pathology of the disease, such as those involving ischemia reperfusion injury (Specific Aim 1), and to initially characterize the pharmacological basis of this effect (Specific Aim 2). The third and final Specific Aim is designed to identify the molecular mechanisms by which aTRA accomplishes cytoprotection, with each sub- aim focusing on a target that has already been identified in preliminary studies as playing an important role in the cytoprotective response. Specifically, those mediators are Nrf2, Grp78, and p38 MAPK, each of which is also a key mediator of ischemia preconditioning. Specific Aim 3 will therefore determine whether (i) aTRA- mediated induction of the anti-oxidant stress response is dependent upon Nrf2; (ii) aTRA-mediated cytoprotection requires the recruitment of the ER (Grp78) mediated stress response pathway; (iii) the recruitment of Nrf2 by aTRA is dependent on p38 MAPK-Grp78 interactions; and (iv) the mechanism(s) of cytoprotection identified in the in vitro model are recapitulated in the in vivo model by testing aTRA induced renoprotection in Nrf2-/- mice. The significance of the current studies resides in their potential to enhance our understanding of retinoid mediated cytoprotection at the molecular and cellular level, which can subsequently provide insights into novel therapeutic strategies effective for clinical interventions during chemical induced tissue injury or hypoxia/ischemia-reperfusion injury.